We have begun to look for distant c-myb transcriptional regulators in the region 25-100 kb upstream region. Because of recent developments in the epigenetics field we can now begin to predict the function of regions of chromatin based upon the presence of histones with specific modifications or specific DNA-binding proteins. Promoter regions and enhancers of activated genes are enriched in histone 3, mono-and tri-methylated on lysine 4 (H3K4me1, H3K4me3) and have acetylated histone 3, for example, H3K9Ac. In an attempt to find regulatory sequences in the upstream region of c-myb, chromatin immunoprecipitation analyzed by microchip (ChIP-on-chip) was carried out using antibodies specific for these marks and a tiling microarray representing the 600kb region surrounding the c-myb gene (40bp spacing). When an antibody specific for trimethyl-H3K4 was used in an experiment performed in myeloid blast cells, M1, it was found that this mark was strongly enriched in the region corresponding to the Mml1 integration site (25kb upstream of c-myb) as well as at the c-myb gene. Subsequent experiment with anti- H3K9, 14Ac antibodies revealed that these regions were also enriched in acetylated histones, confirming that both of these peaks are associated with transcriptional activity. Interestingly, abundance of H3K4me3 at both of these regions was decreased in M1 cells following IL-6-induced differentiation, which suggests that activity of the region corresponding to Mml1 integration is correlated with the transcriptional state of c-myb gene. Changes in H3K27me3 and H3K9me, reported to be associated with transcriptional repression states were not found to largely differ. Next, ChIP-on-Chip with anti-H3K4me3 was performed in the cells lines carrying retrovirus in the Mml1 region. It was shown previously that the steady-state level of c-Myb mRNA and protein were the same or increased in these cell lines compared to M1 cells. Interestingly, the ChIP-on-chip experiment revealed that the H3K4me3 has a different pattern of distribution at c-myb in 3 Mml1 cell lines. The H3K4me3 signal in these cells spread dramatically toward the 3 end of c-myb and the signal was stronger. This data indicated that virus integration at the Mml 1 region results in predominant spreading of the active transcription-related histone mark 25 kb down-stream at the c-myb locus. Monomethylated-H3K4 (H3K4me1) was detected in M1 cells at Mml1 and Mml2 indicating the presence of enhancers and these marks decreased upon differentiation-induced c-myb down-regulation. We also noted that in tumor cells having Mml1 and Mml3 integrations there was a general spreading of the mark throughout the c-myb upstream region including all of Mml regions. In addition there was a specific high peak at the Mml3 integration site in the tumor with the integration. Since the above observations suggest that the Mml1, and Mml2 regions are associated with the transcriptional activity of the c-myb promoter, we decided to look for evidence of physical interaction of this and other regions upstream of c-myb with the promoter of the gene. Using a Chromatin Conformation Capture (3C, chromatin looping) assay we have been able to show that several regions upstream of c-myb interact with the c-myb promoter. This provided the most exciting data, because all three Mml integration regions (Mml1, Mml2, and Mml3) were located at the peaks of predominant interaction loops detected in M1 cells. Two of these interactions were weaker when c-myb was down-regulated by IL-6 in M1 cells. Since CTCF has been shown by many others to be involved in facilitating looping, we carried out ChIP-on-Chip to look for locations of CTCF binding in M1 cell DNA. Prominent CTCF binding sites were discovered at each of the three retrovirus integration sites, Mml1, Mml2 and Mml3 which, as mentioned above, were found at the interaction peaks. Our data indicates that retrovirus integrated upstream of c-myb are brought to close proximity of the c-myb promoter by three dimensional looping. Studies are presently being performed to confirm the presence of the interaction loops in tumor cells.